Pulmonary hypoplasia or lung immaturity is found in as many as 15-20% of all neonatal autopsies. The ability to grow functional 3-dimensional lung tissue from cultured cells for eventual transplantation would be invaluable in preventing the neonatal mortality associated with pulmonary hypoplasia. The long term goal of this project is to alleviate neonatal pulmonary hypoplasia by augmenting damaged lung with in vitro engineered pulmonary tissue constructs. In order to achieve this goal, murine fetal pulmonary cells will be isolated, and utilizing engineered nano-based scaffolds, composite gels, growth factor enhanced media and an optimized microperfusion system, 3-dimensional lung constructs will be formed. The hypothesis of this project is that in an engineered micro-environment, cultured cells will be able to achieve cell to cell contact, adhere, proliferate, and differentiate into macroscopic 3-dimensional tissue constructs. The short term goal of this project is to systematically determine the parameters of a system for engineering lung tissue with appropriate morphology and cytodifferentiation. The specific aims of this project are to: 1- Optimize methods for isolating and culturing heterotypic populations of pulmonary fetal cells using enhanced culture media containing tissue-specific growth factors. 2- To facilitate adhesion, survival, growth and differentiation of cultured fetal lung cells over a prolonged period of time by growing these cells on organotypic 3-dimensional matrices composed of either composite gels or nanofibers electrospun from extracellular matrix derived molecules. 3- Utilize a microperfusion system to enable the continuous bathing of these 3-D constructs with nutrients as well as subject them to pulsatile flow induced shear forces. Morphology and cytodifferentiation of the ensuing 3-D contructs will be evaluated by histology, immunohistochemistry, electron microscopy and biochemical/ molecular biological techniques. The goal of this R-21 application is to explore and optimize the feasibility of this approach and to generate preliminary data for submission of a traditional R01 application. [unreadable] [unreadable]